Physiological characteristics: Oxidative and fermentation tests

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Physiological characteristics Oxidative and
fermentation tests

• Lab 8
• Medgar Evers College
• Prof. Santos

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Metabolism

• Metabolism is the sum total of all chemical
  reactions that occur in a cell.
• 2 types of metabolic pathways catabolism and
  anabolism.

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catabolism

• The breakdown of food material either through
  respiration or fermentation

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Anabolism

• The biosynthesis of materials needed by the cell
  such protein production or photosynthesis

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Laboratory Aim today!

• To determine if our unknown sample carries out
  respiration (oxidative) or fermentation
  (fermentative).
• If our organism is fermentative, which sugars are
  specifically fermented?

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O/F Glucose test

• This test will be done to determine if our
  organism is oxidative or fermentative!
• O/F Glucose is a differential medium that
  contains three important ingredients
• 1- glucose
• 2- peptone
• 3- brom-thymol blue pH indicator that turns
  yellow at acidic pH

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Procedure

• 1- you will inoculate 2 tubes with your unknown
  by stabbing the agar.
• 2- to one tube, you will aseptically layer with a
  couple of drops of mineral oil. This prevents the
  diffusion of oxygen and helps establish anaerobic
  condition.

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• You will repeat the procedure with your two
  control organisms E coli (facultative) and
  Pseudomonas (oxidative)
• A color change from green to yellow means a
  positive test. Due to pH indicator changing as a
  result of slight acid production due to
  respiration. Look for yellow near the top of tube
  if organism is aerobic!

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results
Aerobic condition Anaerobic condition interpretation
Unknown?
Pseudomonas yellow green Strict aerobe
E. coli yellow yellow facultative
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Fermentative tests

• 1- specific sugar tests such as glucose, mannitol
  and lactose.
• 2- Mixed acid fermentation using MR-VP medium
• 3- 2,3- butanediol fermentation
• 4- citrate fermentation

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Specific sugar fermentation reactions

• 1- we will use specific sugar broth with a Durham
  tube inside.
• 2-The pH indicator phenol red will be used to
  detect the formation of acid that indicates
  fermentation.
• 3- you will inoculate your unknown into a tube of
  glucose, lactose and mannitol!
• 4- the control tube will be E.coli into a glucose
  tube.

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Mixed acid fermentation

• Some species of bacteria such as Escherichia,
  Proteus, Salmonella, and Aeromonas can ferment
  glucose and produce acidic products such a lactic
  acid, succinic, acetic, and formic acid.
• In addition they can produce CO2 and H2 gas due
  to the presence of an enzyme called formic
  hydrogenlyase.

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• We will use the medium called MR-VP to inoculate
  our unknown and your control which is E. coli.
• MR-VP medium contains glucose, peptone, and
  dipotassium phosphate.
• Next week we will use methyl red to detect the
  presence of mixed acids!

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2,3- Butanediol fermentation

• Some species of bacteria such as Enterobacter and
  Serratia can ferment glucose and produce a
  neutral end product such as 2,3-butanediol.
• This is important because usually if a type of
  bacteria is for mixed acid it is for neutral
  fermentation. It allows clinical microbiologists
  to distinguish between important gram bacteria.

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• You will inoculate a tube of MR-VP medium with
  your unknown and the control organism which is
  Enterobacter aerogenes.
• Next week, we will discuss how we detect the
  neutral end product using Barritts reagent.

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Citrate fermentation

• Some bacteria can break down citrate to
  oxaloacetate and pyruvate. These intermediates
  are then fermented to produce formate, lactate,
  acetate, acetoin, and carbon dioxide.

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• We will use Simmons citrate agar. It contains
  citrate and ammonium salts to serve as a source
  of nitrogen. As the organisms break down the
  citrate they must also break down the ammonium
  salts into ammonia. Ammonia raises the pH and the
  agar changes color from green to Prussian blue.

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• You will inoculate the Simmons citrate agar with
  your unknown and the control which is
  Enterobacter aerogenes. First streak with a
  needle and then stab it!

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Oxidative tests

• 1- cytochrome oxidase test
• 2- catalase test
• 3- nitrate reduction

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Cytochrome C oxidase test

• Aerobic organisms such as Pseudomonas carry out
  respiration and possess an enzyme called
  cytochrome oxidase that catalyzes the transfer of
  electrons from reduced cytochrome c to molecular
  oxygen.
• You will streak half a TSA plate with your
  unknown and the other half with your control
  Pseudomonas aeruginosa.

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• Next week, we will use an artificial electron
  acceptor to detect the enzyme! The artificial
  acceptor is called N,N,N,N-tetramethyl-p-phenylene
  diamine!

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Catalase test

• A dangerous by product of aerobic respiration is
  hydrogen peroxide. Aerobes produce an enzyme
  called Catalase that breaks down the hydrogen
  peroxide into water and oxygen.
• Next week, you will use hydrogen peroxide to
  detect the presence of the enzyme by your
  unknown. The TSA plate will also be used for this
  experiment. The control is a nutrient agar slant
  inoculated with S. aureus.

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Nitrate reduction

• Some bacteria can use nitrates as the final
  electron acceptor instead of oxygen.
• Some bacteria such as Bacillus can reduce the
  nitrates to gas end products such as N2O or N2.
• Others such as E.coli can reduce the nitrates to
  nitrites.

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• We will assay for the final end product, gas or
  nitrite!
• We will use beef extract medium with potassium
  nitrate. A Durham tube will be in place to detect
  gas formation.
• You will inoculate a tube of beef extract with
  your unknown and the control E. coli.

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• Next week, we will detect gas formation by
  looking at Durham tube and detect nitrite
  formation using Nitrate reagent A and B. These 2
  reagents react with nitrite to give you a deep
  red color!